1. Field of the Invention
The present invention relates to a method detecting ceruloplasmin levels in urine samples for pregnancy screening in animals. More particularly, the present invention disclosed a method to identify pseudopregnancy, pregnancy, and pregnancy loss using an acute phase protein ceruloplasmin as a non-invasive marker.
2. Description of the Related Art
Ceruloplasmin (Cp) is a multicopper oxidase enzyme involved in the safe handling of oxygen in some metabolic pathways of vertebrates. Discovered in 1948, a blue protein from the a2-giobulin fraction of human serum possessing oxidase activity towards aromatic diamines and catechol was purified by Holmberg and Laurell (Homberg and Laurell, 1948). It was denoted ceruloplasmin, literally meaning ‘a blue substance from plasma’. Specialized copper sites have been recruited during evolution to provide long-range electron transfer reactivity and oxygen binding and activation in proteins destined to cope with oxygen reactivity in different organisms. Ceruloplasmin belongs to the family of multicopper oxidases which are among the few enzymes able to bind molecular oxygen to perform its complete reduction to water (Malmstrom B G, 1982; Farver O. and Pecht I., 1997). Ceruloplasmin contains 95% of the copper in serum (Harris and Gitlin, 1996). Cp found in serum is expressed in the liver, but it is also expressed in the brain, lung, spleen and testis. Aceruloplasminaemia is an autosomal recessive disorder of iron metabolism characterized by the complete absence of ceruloplasmin (Yoshida et al., 1995; Harris et al., 1995). The role of Cp in tissue iron overload and the subsequent clinical findings of diabetes, retinal degeneration and neurodegeneration has been associated with iron overload in aceruloplasminaemic patients (Takahashi et al., 1996). Thus it is clearly indicated that ceruloplasmin plays an essential role in iron metabolism. Evaluation of Ceruloplasmin Activity is commonly used for diagnosis of copper metabolism disorders in humans such as Wilson's disease due to the high affinity of copper for the ceruloplasmin protein (Macintyre et al, 2004; Merle et al., 2009; Sunderman and Nomoto, 1970).
Ceruloplasmin is also associated with reproduction. Copper-deficient female rats seem to be protected against mortality. This protection has been suggested to be provided by estrogens, since estrogens alter the subcellular distribution of copper in the liver, an increase in plasma copper levels and subsequent ceruloplasmin synthesis (Fields et al., 1986). The cause of elevated ceruloplasmin during pregnancy is currently unknown but may related to role of ceruloplasmin in the immunologic recognition of the fetus, inflammatory responses occurring during pregnancy, iron homeostasis, angiogenesis, and/or oxidant defenses. Enzymatic assays for the determination of ceruloplasmin oxidase activity have been described to have greater biological relevance than determination of total ceruloplasmin protein concentration by immulogic techniques that can report ceruloplasmin protein with no enzymatic activity (Macintyre et al, 2004; Merle et at., 2009).
As mentioned previously, ceruloplasmin is the major copper-carrying protein in the blood serum. Its primary clinical function is to detect Wilson's Disease that is an inherited disorder in which there is too much copper in the body's tissues. U.S. Pat. No. 7,407,743 disclosed an assay to measure ceruloplasmin concentration on a blood spot in order to screen Wilson's Disease in population. Although serum levels of some acute phase proteins typically associated with the immune system and inflammation were found to increase during pregnancy in mammals such as humans and dogs (Vannucchi, 2002; Markowitz 1955; Burrows 1971; Ulntas 2009), daily and even weekly blood collections from mammals, especially endangered/exotic species, are challenging to obtain. For example, it is difficult to collect blood samples regularly in giant pandas and polar bears. Therefore, it is necessary to find alternative method to detect pregnancy non-invasively in animal urine samples. In addition, it is also necessary to use urine non-invasive method to distinguish between pregnancy and pseudopregnancy in animals.